Abstract:

A method for using inactivated Japanese encephalitis virus particles as
an adjuvant of a vaccine is provided. A method for using inactivated
Japanese encephalitis virus (JEV) particles as an adjuvant of various
vaccines or a mixed vaccine, said JEV particles being obtained by
inoculating JEV Beijing-1 strain to Vero cells, culturing said
JEV-infected cells to give cultured cells or culture supernatant,
purifying JEV particles from said cultured cells or culture supernatant
and inactivating said JEV particles with formalin, a method for preparing
a (mixed) vaccine which comprises a step of letting inactivated Japanese
encephalitis virus be contained, and a mixed vaccine prepared by said
method.

Claims:

1-4. (canceled)

5. A method for preparing a vaccine, the method comprising: letting
inactivated Japanese encephalitis virus, obtained by culture of Vero
cells, be comprised in the vaccine as an adjuvant.

6. (canceled)

7. The method of claim 5, wherein the inactivated Japanese encephalitis
virus has a particle structure.

8. The method of claim 5, wherein the letting is of adding the
inactivated Japanese encephalitis virus to a vaccine.

9. The method of claim 5, wherein the vaccine is a mixed vaccine in which
two or more different vaccines are mixed together.

10. The method of claim 9, wherein said vaccine is selected from the
group consisting of a diphtheria vaccine, a pertussis vaccine, a tetanus
vaccine, a polio vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a
rabies vaccine, a measles vaccine, a rubella vaccine, an influenza
vaccine, a mumps vaccine, a varicella vaccine, a rota vaccine, a smallpox
vaccine, a yellow fever vaccine, a mite-mediated encephalitis vaccine, an
Hib vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a
pneumococcus vaccine and a vaccine against meningitis caused by Neisseria
meningitidis.

11. The method of claim 5, wherein the inactivated Japanese encephalitis
virus is in particles which are present in a concentration of 250 ng to
34 μg/mL.

12. A vaccine prepared by the method of claim 5.

13. The vaccine of claim 12, further comprising an aluminum adjuvant.

14. The method of claim 7, wherein the letting is adding the inactivated
Japanese encephalitis virus to a vaccine.

15. The method of claim 7, wherein the vaccine is a mixed vaccine in
which two or more different vaccines are mixed together.

16. The method of claim 8, wherein the vaccine is a mixed vaccine in
which two or more different vaccines are mixed together.

17. The method of claim 14, wherein the vaccine is a mixed vaccine in
which two or more different vaccines are mixed together.

18. The method of claim 15, wherein said vaccine is selected from the
group consisting of a diphtheria vaccine, a pertussis vaccine, a tetanus
vaccine, a polio vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a
rabies vaccine, a measles vaccine, a rubella vaccine, an influenza
vaccine, a mumps vaccine, a varicella vaccine, a rota vaccine, a smallpox
vaccine, a yellow fever vaccine, a mite-mediated encephalitis vaccine, an
Hib vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a
pneumococcus vaccine and a vaccine against meningitis caused by Neisseria
meningitidis.

19. The method of claim 16, wherein said vaccine is selected from the
group consisting of a diphtheria vaccine, a pertussis vaccine, a tetanus
vaccine, a polio vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a
rabies vaccine, a measles vaccine, a rubella vaccine, an influenza
vaccine, a mumps vaccine, a varicella vaccine, a rota vaccine, a smallpox
vaccine, a yellow fever vaccine, a mite-mediated encephalitis vaccine, an
Hib vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a
pneumococcus vaccine and a vaccine against meningitis caused by Neisseria
meningitidis.

20. The method of claim 17, wherein said vaccine is selected from the
group consisting of a diphtheria vaccine, a pertussis vaccine, a tetanus
vaccine, a polio vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a
rabies vaccine, a measles vaccine, a rubella vaccine, an influenza
vaccine, a mumps vaccine, a varicella vaccine, a rota vaccine, a smallpox
vaccine, a yellow fever vaccine, a mite-mediated encephalitis vaccine, an
Hib vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a
pneumococcus vaccine and a vaccine against meningitis caused by Neisseria
meningitidis.

21. The method of claim 7, wherein the inactivated Japanese encephalitis
virus is comprised in a concentration of 250 ng to 34 μg/mL.

22. The method of claim 8, wherein the inactivated Japanese encephalitis
virus is in particles which are comprised in a concentration of 250 ng to
34 μg/mL.

23. The method of claim 14, wherein the inactivated Japanese encephalitis
virus is comprised in a concentration of 250 ng to 34 μg/mL.

24. The method of claim 15, wherein the inactivated Japanese encephalitis
virus is comprised in a concentration of 250 ng to 34 μg/mL.

25. The method of claim 16, wherein the inactivated Japanese encephalitis
virus is in particles which are comprised in a concentration of 250 ng to
34 μg/mL.

26. A vaccine prepared by the method of claim 11.

27. The vaccine of claim 26, further comprising an aluminum adjuvant.

Description:

TECHNICAL FIELD

[0001] The present invention relates to a method for using inactivated
Japanese encephalitis virus (hereinafter also referred to as "JEV")
particles as an adjuvant for various mixed vaccines or polyvalent
vaccines.

BACKGROUND ART

[0002] Vaccination to infectious diseases should preferably be finished by
infancy. For this purpose, many kinds of vaccines need to be inoculated
in a fixed period of time. As a devise for efficient vaccination to
various infectious diseases, mixed vaccines in which several vaccines are
mixed together have been developed. A mixed vaccine has many merits
including: (i) it may provide protection to plural pathogens
simultaneously, (ii) it may eliminate troublesomeness of inoculation
schedule of plural vaccinations, (iii) it may reduce a cost for
vaccinations (technical fee) and burden of time for individuals who
receive vaccinations with increase in a vaccination rate being expected,
(iv) it may reduce burden of medical staff, and (v) it may reduce burden
to environments by diminution of waste materials through reduction of
vaccination frequency. Thus, for a mixed vaccine, the more vaccine
antigens are contained therein, the better.

[0003] However, the number of vaccine antigens to be mixed together for
preparing a mixed vaccine is limited. Namely, the larger number of
vaccine antigens are mixed, the less an amount of each of the vaccine
antigens can be administered. Besides, some kinds of antigens may
interfere with each other to thereby lead to reduction of their
antibody-inducing capacity, which may result in reduction in antibody
titer of a vaccine and make efficient protection against infection
difficult.

[0004] Concrete mixed vaccines that have been published up till the
present include a diphtheria/pertussis/tetanus (DPT) mixed vaccine
preparation "VACCINE COMPOSITION" (cf. e.g. Patent reference 1), a mixed
vaccine containing papilloma virus (HPV) antigen "Novel Composition" (cf.
e.g. Patent reference 2), a polyvalent DPT polio vaccine "MULTIVALENT
DTP-POLIO VACCINES" (cf. e.g. Patent reference 3), a method for inducing
cellular immune activity of a live vaccine in an inactivated vaccine and
a mixed vaccine obtained by said method (cf. e.g. Patent reference 4),
and the like. In addition to these, mixed vaccines of numerous
combinations have already been published. For a Japanese encephalitis
virus vaccine, mixed vaccines with DPT vaccine, hepatitis B vaccine
(HepB), hepatitis A vaccine (HepA), and the like have been reported (cf.
e.g. Non-patent reference 1). Development of a mixed vaccine with more
combinations is desired but there is the problem of reduced effect of a
vaccine as described above. In order to solve the problem, development of
such an adjuvant that allows for a higher antibody titer with as little
amount of an antigen as possible is desired.

[0005] In general, an adjuvant, when added to a vaccine antigen, is known
to enhance immunogenicity of the vaccine antigen and includes an aluminum
gel particle (aluminum salt), an oil adjuvant comprising a mineral oil as
a main component, a surfactant-like adjuvant such as saponin purified
from white hyacinth bean, and a TH1-inducing adjuvant derived from
intracellular toxins (LPS, etc.). For an adjuvant for use in a vaccine
for animal, an oil adjuvant may be often used which is locally more
reactive than an aluminum adjuvant. For an adjuvant to be used in a
vaccine for human, however, safety is required in addition to efficacy.

[0006] For an adjuvant to be used in a vaccine for human, an aluminum gel
has hitherto been used primarily as its efficacy has been proved but
recently newly developed MPL and double-stranded RNA have got to be
chosen for use. Already known adjuvant compositions include "Vaccine"
consisting of an immunostimulating agent (MPL) and a metal salt (cf. e.g.
Patent reference 5), an oil-in-water emulsion "Adjuvant composition" (cf.
e.g. Patent reference 6) which comprises oil such as 3D-MPL, squalene,
alpha-tocopherol, polyoxyethylene sorbitan monooleate, and the like, a
synthetic compound adjuvant "Immunologic adjuvant compound" (cf. e.g.
Patent reference 7), "Vaccine preparation" (cf. e.g. Patent reference 8)
which utilizes cholera toxin, "Use of virus-like particle as adjuvant"
(cf. e.g. Patent reference 9) which utilizes virus-like particle (VLP)
formed from a particle-forming polypeptide of a surface antigen of
hepatitis virus, and many others.

[0021] As described above, while development of a mixed vaccine is desired
as enabling immunization to more infectious diseases at a stretch, the
larger number of vaccine antigens are mixed, the less an amount of each
of the vaccine antigens is forced. Thus, in order to make up for this
drawback, development of such an adjuvant that allows for a higher
antibody titer even with a lower amount of an antigen is desired.

Means for Solving the Problems

[0022] Under the circumstances, the present inventors have earnestly
continued research activities and as a result have found that, by adding
inactivated JEV particles to a vaccine solution containing all or a
portion of protective antigens to infectious diseases, i.e. diphtheria,
pertussis, tetanus, polio, hepatitis B and hepatitis A, immunization was
induced at a higher level and more rapidly as compared to the
corresponding vaccine solution not containing said inactivated JEV
particles, namely, that the inactivated JEV particles do possess the
activity as an adjuvant, and thus completed the present invention. Up
till the present invention, there was no idea to utilize the adjuvant
activity exerted by the inactivated JEV particles per se, the inactivated
JEV particles being a vaccine antigen of a mixed vaccine. The idea the
present inventors have firstly found out. An object of the present
invention is to provide a method for using inactivated JEV particle as an
adjuvant for various mixed vaccines and a mixed vaccine containing the
JEV.

[0023] Accordingly, the present invention is as follows:

(1) A method for using inactivated Japanese encephalitis virus as an
adjuvant of a vaccine. (2) The method of (1) above wherein the
inactivated Japanese encephalitis virus is one obtained by inactivating
Japanese encephalitis virus obtained by cell culture. (3) The method of
(1) or (2) above wherein the inactivated Japanese encephalitis virus has
a particle-like structure. (4) The method of any one of (1) to (3) above
wherein the vaccine is a mixed vaccine. (5) A method for preparing a
vaccine which comprises a step of letting inactivated Japanese
encephalitis virus be contained as an adjuvant. (6) The method of (5)
above wherein the inactivated Japanese encephalitis virus is one obtained
by inactivating Japanese encephalitis virus obtained by cell culture. (7)
The method of (5) or (6) above wherein the inactivated Japanese
encephalitis virus has a particle-like structure. (8) The method of any
one of (5) to (7) above wherein said step is a step of adding the
inactivated Japanese encephalitis virus to a vaccine. (9) The method of
any one of (5) to (8) above wherein the vaccine is a mixed vaccine in
which two or more different vaccines are mixed together. (10) The method
of (9) above wherein said vaccine is selected from the group consisting
of a diphtheria vaccine, a pertussis vaccine, a tetanus vaccine, a polio
vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a rabies vaccine,
a measles vaccine, a rubella vaccine, an influenza vaccine, a mumps
vaccine, a varicella vaccine, a rota vaccine, a smallpox vaccine, a
yellow fever vaccine, a mite-mediated encephalitis vaccine, an Hib
vaccine, a typhoid vaccine, a cholera vaccine, a BCG vaccine, a
pneumococcus vaccine and a vaccine against meningitis caused by Neisseria
meningitidis. (11) The method of any one of (5) to (10) above wherein
the inactivated Japanese encephalitis virus particles are at 250 ng to 34
μg/mL. (12) A vaccine prepared by the method of any one of (5) to (11)
above. (13) The vaccine of (12) above which comprises an aluminum
adjuvant.

MORE EFFICACIOUS EFFECTS THAN PRIOR ART

[0024] According to the present invention, a method for preparing a
vaccine comprising inactivated JEV particles as an adjuvant and a vaccine
prepared by said method are provided. For instance, by adding inactivated
JEV particles to a mixed vaccine as an adjuvant, immunization may be
induced at a higher level and more rapidly as compared to the
corresponding vaccine with no addition of said inactivated JEV particles.
Namely, since a given antibody titer capable of attaining protection
against infection may be obtained with a smaller amount of an antigen, an
amount of each of antigens to be contained in a mixed vaccine may be
reduced to thereby allow for more kinds of antigens be contained in a
mixed vaccine.

[0025] Besides, in accordance with the method of the present invention,
not only immunization of a mixed vaccine may be exerted but also an
antibody titer for preventing infection of Japanese encephalitis virus
may be increased. Thus the mixed vaccine of the invention is also used as
a Japanese encephalitis vaccine.

[0030] FIG. 5 shows the results of ELISA carried out on sera from mice
immunized with a mixture of a HB vaccine and JEV particles (Anti-HBs
antibody titer).

[0031] FIG. 6 shows the results of ELISA carried out on sera from mice
immunized with a mixture of an HepA vaccine and JEV particles (Anti-HVA
antibody titer).

BEST MODE FOR CARRYING OUT THE INVENTION

[0032] The present invention is characterized by a method for preparing a
vaccine which comprises a step of adding inactivated JEV as an adjuvant.

[0033] JEV strains for use herein may be any with no specific limitation.
Beijin-1 strain was used for the invention. JEV may be obtained by two
methods as described hereinbelow. One is a method where JEV is inoculated
in the brain of mice and the viruses are propagated in the brain and then
purified therefrom. The other is a method where JEV is inoculated in
culture cells, the virus-infected cells are cultured and the viruses are
purified from the grown JEV-infected cells and culture supernatant. From
the viewpoint of animal protection, the method with propagation with the
culture cells is preferable. For a host cell for propagation of JEV, Vero
cells may be used that allows for good propagation of JEV and production
of an antigen with higher antigenicity. A culture medium for expansion
culture of cells may suitably be selected from one commonly used for
tissue culture such as M199 medium, Eagle MEM medium, and the like,
preferably Dulbecco MEM medium optionally supplemented with an amino
acid, salts, an anti-mold/anti-bacterial agent, animal serum, and the
like. A high-density culture using a microcarrier may sometimes be
carried out for obtaining a large amount of JEV particles. A number of
microcarriers for this purpose are known. A microcarrier suitable for
growth of Vero cells includes Cytodex (Cytodex I, Amersham Pharmacia
Biotech) at a concentration of 5 g/L or less.

[0034] Culture temperature and duration of culture may be adjusted
depending on a combination of the cell types, an amount of viruses
inoculated, a scale and procedure of culture, and the like. For instance,
when JEV is propagated by static culture or roller bottle culture with
Vero cells, the cells may be cultured in a growth medium consisting of
Dulbecco MEM medium supplemented with a non-essential amino acid and
bovine serum at a culture temperature of 32° C. to 38° C.
for duration of culture of 2 to 7 days. For propagation of JEV, the
culture medium is removed by aspiration when the growth reached stable
stage and, after washing with a phosphate buffer/saline several times,
JEV is inoculated to the cells at multiplicity of infection (M.O.I) of
0.01 to 0.0001. For a maintenance medium used after viral inoculation, a
serum-free, low-protein level medium may preferably be used. For
instance, VP-SFM (GIBCO) supplemented with L-glutamic acid may be used.
The cells may be cultured at the culture temperature as described above
for 4 to 7 days. After completion of culture, the resultant culture, i.e.
cell-breakage solution or culture supernatant, may be recovered, added
with formalin and left to stand at around 4° C. for 1 to 3 months
or more to inactivate the viruses.

[0035] For purification of JEV particles from the obtained JEV-containing
solution, purification procedures commonly used in protein chemistry may
be used, e.g. centrifugation, salting-out, normal filtration,
ultrafiltration, isoelectric precipitation, electrophoresis, ion exchange
chromatography, gel filtration chromatography, affinity chromatography,
hydrophobic chromatography, hydroxyapatite chromatography, and the like.
Among these, suitable procedure may be selected and used in combination
thereof. In the Examples herein, JEV particles were purified by
subjecting the JEV-containing solution to sucrose-gradient centrifuge,
fractionating and pooling fractions of JEV particles, and performing
affinity chromatography with cellulose sulfate ester gel. The solution
containing the inactivated JEV after purification may be diluted with
e.g. a phosphate buffer or saline to a viral content suitable for use as
an adjuvant. The thus obtained solution containing the inactivated JEV
particles may be added to various vaccines as an adjuvant.

[0036] The method of the present invention may be used for either a
vaccine against viral infectious diseases or for a vaccine against
bacterial infectious diseases. A vaccine against viral infectious
diseases includes, for instance, a vaccine against hepatitis A, hepatitis
B, rabies, polio, measles, rubella, influenza, yellow fever,
mite-mediated encephalitis, mumps, varicella, rota, smallpox, and the
like. A vaccine against bacterial infectious diseases includes, for
instance, pertussis, diphtheria, tetanus, typhoid, cholera, meningitis
caused by Neisseria meningitidis, Hib (Haemophilus influenzae type b),
BCG, pneumococcus, and the like. A vaccine selected from these may be
used solely or may be a mixed vaccine in which two or more vaccines are
mixed together. However, since an amount of an antigen to be contained in
a vaccine may be reduced when inactivated JEV particles are used as an
adjuvant, the vaccine may preferably be used as a mixed vaccine where an
amount of antigens is restricted.

[0037] Dosage form may be injections, syringes, transdermal agents, and
sprays, with the vaccine being a liquid preparation in which
liquid-liquid products have already been mixed in a bulking step, a
systemic preparation in which a liquid-liquid product and a
liquid-lyophilized product are put in containers for one-touch mixing
when use, or a preparation in which a liquid-liquid product and a
liquid-lyophilized product are mixed manually when use. A route of
administration includes intramuscular injection, subcutaneous injection,
transdermal administration, and the like, which may suitably be selected
depending on purposes. A route of administration may also be such that
either the same site of administration or different sites of
administration may be used.

[0038] For the vaccines described above, a suitable adjuvant may be added
to an antigen solution for enhancing immunogenicity. An amount of an
adjuvant to be added may suitably be set depending on the kind, the
number and an amount of an antigen. The kind of an adjuvant includes
aluminum hydroxide gel, aluminum phosphate gel, aluminum sulfate gel,
mineral oil, non-mineral oil, and the like. An aluminum gel, which has
been used for a number of vaccine for human and well proved to be safe,
is mostly used. Although an aluminum gel is proved to be safe, if its
dose is not observed, adverse side effects such as aluminum
encephalopathy or aluminum bone disease may be seen. Therefore, an
aluminum gel may preferably be used at as low an amount as possible.
Since the inactivated JEV particles in accordance with the present
invention allow for reduction of an amount of an aluminum gel in an
aluminum-containing vaccine, the vaccine of the present invention may
effectively be used in combination with an aluminum gel.

[0039] The inactivated JEV particles may be used at a range of 250 ng to
34 μg/mL where the adjuvant activity is exerted, preferably at 1 to 16
μg/mL, more preferably at 2 to 8 μg/mL. For schedule of addition of
the inactivated JEV particles to a vaccine, the inactivated JEV particles
may be added while or after said vaccine is prepared. For instance, a
suitable amount of the inactivated JEV particles may be added to a stock
solution of an attenuated live vaccine or a stock solution of an
inactivated vaccine or a mixture thereof and then the solution may
suitably be diluted with a phosphate buffer or saline. Before addition of
the inactivated JEV particles, an aluminum gel may be used as occasion
demands. The vaccine and an aluminum gel may be used at a range of 50 ng
to 80 μg/mL and at 100 to 400 μg/mL, respectively.

[0040] Evaluation of the inactivated JEV particles as an adjuvant may be
done by comparing immune response induced when a vaccine or a mixed
vaccine containing the inactivated JEV particles is injected to animals
via subcutaneous, intramuscular or intradermal administration with immune
response induced when the vaccine not containing the inactivated JEV
particles is injected to the animals. For the evaluation, a small animal
such as rat, mouse, guinea pig or rabbit may be used. For instance,
animals immunized with a vaccine containing the inactivated JEV particles
may be bled, serum be isolated therefrom and an antibody (titer) in the
obtained serum be measured for comparison with an antibody (titer) in
serum from animals immunized with a vaccine not containing the
inactivated JEV particles to thereby evaluate the inactivated JEV
particles as an adjuvant. A method for measuring an antibody (titer)
includes ELISA, EIA, a neutralization test with a toxin and cell line,
and the like, any of which may be used.

[0041] The present invention is explained in more detail by means of the
following Reference Examples and Examples but should not be construed to
be limited thereto.

Reference Example 1

[0042] Japanese encephalitis virus particles were prepared as described in
Japanese patent publication No. 2000-83657. Briefly, Japanese
encephalitis virus (Beijin-1) at M.O.I. of 0.01 was inoculated to Vero
cells grown by suspension culture with Dulbecco MEM medium and, after
absorption at 37° C. for 90 minutes, the cells were cultured at
the same temperature for 3 to 5 days while supplementing the medium. The
culture solution was subjected to a sucrose-gradient centrifuge to
recover viral fractions and the virus particles were purified by
cellulose sulfate ester gel. For inactivation of the virus particles, the
virus particles were left to stand under conditions of 0.08 vol %
formalin in a refrigerator for about half a year. The thus obtained
inactivated Japanese encephalitis virus particles were used in Examples.
An amount of protein in the inactivated Japanese encephalitis virus
particles was measured by Lowry method.

Reference Example 2

(1) Elisa Used for Measurement of Antibody Titer

[0043] For an immobilized antigen, pertussis toxoid (PT) at 2.5 μg/mL,
diphtheria toxoid (DT) at 5 μg/mL, tetanus toxoid (TT) at 5 μg/mL,
hepatitis B virus surface antigen (HBs) at 5 μg/mL, poliovirus type 1
at 1.9 Du/mL, poliovirus type 2 at 0.975 Du/ml, poliovirus type 3 at 1.5
Du/mL, and inactivated JEV particles at 5 μg/mL. For HBs and
inactivated JEV particles, an amount of protein was measured by Lowry
method. For each of DPT, an amount of protein nitrogen was measured by
Kjeldahl method and converted to an amount of protein. For poliovirus, an
amount of D antigen was measured. An antigen to be immobilized was added
to a 96-well plate (Nunc, Maxisorp) at 100 μL/well and the plate was
left to stand at 4° C. overnight for immobilization. On the next
day, each well was washed three times with 350 μL of PBS containing
0.05% Tween 20 (PEST) and added with 350 μL/well of Block Ace
(Dainippon Sumitomo Pharma Co., Ltd., hereinafter abbreviated to "BA")
diluted 4-fold with PBS and the plate was left to stand at room
temperature for 2 hours. After 2 hours, 4-fold diluted BA was removed
sufficiently and the wells were washed three times with 350 μL/well of
PBST. Then, samples were diluted with BA diluted 10-fold with PBS
containing 0.05% Tween 20 and each 100 μL/well of the diluted samples
was added. After reaction at 37° C. for 2 hours, the wells were
washed three times with 350 μL/well of PBST. After the washing
solution was removed sufficiently, each 100 μL/well of HRP-labeled
anti-mouse IgG goat antibody (American Qualax, A131PS) or HRP-labeled
anti-mouse IgG rat antibody (Zymed, 04-6020), HRP-labeled anti-rat IgG
(H+L) goat antibody (Zymed, 81-9520), each diluted 2000-fold with the
solution used for dilution of the samples, was added to the wells for
reaction at 37° C. for 1 hour. After 1 hour, the solution was
discarded sufficiently and the wells were washed with 350 μL/well of
PBST four times and with the same amount of distilled water twice. A
chromogenic substrate TMB+(Dako) was added at 100 μL/well for reaction
under shading at room temperature for 30 minutes. Then, 1 N sulfuric acid
was added at 100 μL/well to stop development and absorbance at 450 nm
was measured.

(2) Preparation of Standard Serum and Calculation of Antibody Titer

[0044] Serum, where a sufficient antibody titer was proved by the antibody
titer measuring system as described below two to four weeks after
immunization of an animal with an antigen for protection against an
infectious disease, was used as standard serum. By way of example,
standard serum to each of DPT antigens was one bled from an animal
immunized for 2 weeks with intraperitoneal administration of 0.5 mL of
the current DPT preparation diluted 5-fold. For an antibody titer of the
respective antigens, sera from 26 to 30 mice not immunized were diluted
50-fold or 200-fold with a dilution solution for samples and each of the
diluted samples in duplicate were subject to ELISA as described above to
measure OD 450 nm. A mean value of the measured OD 450 nm plus two times
standard deviation was set for a cut-off. The standard sera were serially
diluted and OD 450 nm was measured. Dilution fold in excess of the
cut-off value (PT: 12,800-fold, DT: 96,000-fold, TT: 320,000-fold, JEV:
1,024,000-fold) was used as an antibody titer of standard serum. Namely,
serum at the dilution fold was set as 1 EU of the respective antibody
titer for measurement of the samples. For ELISA of anti-poliovirus
antibody, a sample diluted 100-fold was used for measurement of OD.

(3) Measurement of Antibody Titer of Anti-HBs Antibody and Determination
of Rate of Change to Positive

[0045] For anti-HBs antibody titer, not only ELISA but also IMx Ausab
Assay System (abbott, 2262-83) were used to measure antibody titer and a
rate of change to positive.

Example 1

Adjuvant Activity of JEV Particles to DPT Vaccine Antigen

[0046] The inactivated Japanese encephalitis virus (JEV) particles
obtained in Reference Example 1 and each antigens of DPT were mixed
together in a composition as shown in Table 1. The obtained mixture (0.5
mL) was inoculated intraperitoneally to SPF mice (C57BL/6s; 4 mice/group)
of 10 weeks old (male). The animals were bled before inoculation and on
Week 2, 3, and 4 after primary inoculation. After bleeding at Week 4,
booster was administered with the same mixture. The animals were bled on
Week 1 and 2 after the second inoculation and antibody titer of the
respective sera was measured in accordance with the method for
measurement of antibody titer as described in Reference Example 2-(1). As
a result, negative interference of antigens to each other due to addition
of JEV was not observed. It was found that an antibody titer to the
respective antigens of DPT (PT, DT, TT) in the group with addition of JEV
was higher than that in the group with DPT alone (FIGS. 1a, b, c). The
adjuvant activity of JEV particles could be seen on Week 2 after primary
inoculation.

[0047] (1) The JEV particles obtained in Reference Example 1 and each
antigens of DPT were mixed together in a composition as shown in Table 2.
The obtained mixture (0.5 mL) was inoculated intraperitoneally to SPF
mice (C57BL/6s; 5 mice/group) of 9 weeks old (male). The animals were
bled before inoculation and on Week 4 after primary inoculation. Antibody
titer of the respective sera was measured in accordance with the method
for measurement of antibody titer as described in Reference Example
2-(1). The outcomes of the measurement of antibody titer are shown in a
geometrical mean. As a result, it was found that an antibody titer to the
respective antigens of DPT (PT, DT, TT) in Groups 2 to 7 was higher than
that in Group 1 where the JEV particles were not added (FIG. 2).

(2) The JEV particles obtained in Reference Example 1 and each antigens
of DPT were mixed together in a composition as shown in Table 3. The
obtained mixture (0.5 mL) was inoculated intraperitoneally to BDF-1 mice
(4 mice/group) of 9 weeks old (female). The animals were bled before
inoculation and on Week 4 after primary inoculation. Antibody titer of
the respective sera was measured in accordance with the method for
measurement of antibody titer as described in Reference Example 2-(1). It
was found that an antibody titer in Groups with addition of the JEV
particles was higher than that in Group with no addition of the JEV
particles (FIG. 3).

[0048] The JEV particles obtained in Reference Example 1 and the
commercially available IPV vaccine (IMOVAX POLIO Sanofi Pasteur) diluted
2-fold and 4-fold were mixed together in a composition as shown in Table
4. The obtained mixture (0.5 mL) was intramuscularly injected to SPF rats
(Wister; 3 rats/group) of 8 weeks old (female) at the thigh of hind leg.
The animals were bled before inoculation and on Week 4 and 8 after
primary inoculation. Antibody titer of the respective sera was measured
in accordance with the method for measurement of antibody titer as
described in Reference Example 2-(1). It was found that an antibody titer
in Groups with addition of the JEV particles was higher than that in
Group with no addition of the JEV particles (FIG. 4).

[0049] The JEV particles obtained in Reference Example 1 and HBs antigen
of HB vaccine were mixed together in a composition as shown in Table 5.
The obtained mixture (1 mL) was inoculated intraperitoneally to BALE/c (5
mice/group) of 5 weeks old (male). The animals were bled before
inoculation and on Week 3, 5 and 6 after primary inoculation. Antibody
titer of the respective sera was measured in accordance with the method
for measurement of antibody titer as described in Reference Example
2-(1). It was found that an antibody titer in Groups with addition of the
JEV particles was higher than that in Group with no addition of the JEV
particles (FIG. 5). When JEV was added to HB vaccine at the dose of 0.25
μg/head, antibody titer was recovered to the same or more level of HB
vaccine at the dose of 0.5 μg/head. Antibody titer of the respective
sera was also measured in accordance with the method as described in
Reference Example 2-(3). Group 4 with addition of the JEV particles
showed a higher rate of change to positive than Group 3 with no addition
of the JEV particles (Table 6).

[0050] The JEV particles obtained in Reference Example 1 and HAV antigen
of HepA vaccine were mixed together in a composition as shown in Table 7.
The obtained mixture (1 mL) was inoculated intraperitoneally to BALE/c (5
animals/group) of 5 weeks old (male). The animals were bled before
inoculation and on Week 2 and 7 after primary inoculation. Antibody titer
of the respective sera was measured by ELISA. For a primary solid phase,
anti-HAV rabbit antiserum was used. After washing three times with PEST,
an HAV antigen was immobilized at 1.25 ng/50 μL/well. Samples were
applied for reaction at 4° C. overnight. After washing three times
with PBST, a secondary antibody with HRP conjugate was reacted. After
washing four times with PEST, a substrate was reacted and absorbance was
measured. The measurement was also done for 30 samples of non-immunized
sera, a cut-off (3SD) was calculated and a rate of change to positive was
obtained (2 w cut-off: 0.061, 7 w cut-off: 0.065).

[0051] It was found that an antibody titer in Groups with addition of the
JEV particles was higher than that in Group with no addition of the JEV
particles (FIG. 6). There was distinction in a rate of change to positive
between Groups 1 and 2 on Week 2 after inoculation and between Groups 3
and 4 on Week 2 and 7 after inoculation (Table 8).